The HIV protease inhibitors as a part of highly active antiretroviral therapy (HAART) have improved the course of HIV disease. However, new clinical complications have emerged as a result of the therapy, including peripheral and coronary arterial diseases, and metabolic disturbances. Our preliminary studies suggest that the HIV protease inhibitors may impair endothelial barrier function, down regulate occludin expression, increase reactive oxygen species (ROS) production, and activate certain type of motigen activated protein kinases (MAPKs). Thus, further investigations are proposed with three specific aims: 1. Determine the effect of HIV protease inhibitors on endothelial permeability. Based upon our preliminary data, we hypothesize that HIV protease inhibitors will increase endothelial cell permeability. Accordingly, we will further characterize in vitro the formation and maintenance of tight intracellular junctions by cultured human coronary artery endothelial cells (HCAECs). Using this system we will evaluate the effects of HIV protease inhibitors on transendothelial electrical resistance (TER) and paracellular flux. Next, using an intact porcine artery perfusion model, we will quantitatively assess changes in endothelial layer permeability by measuring gold particle transport into the vessel wall and modeling of these results. 2. Assess the effects of HIV protease inhibitors on endothelial tight junction structures and gene expression. Specifically, we propose that HIV protease inhibitors alter gene expression of the tight junction molecules occludin, ZO-1, and actin. These hypotheses are supported by our preliminary findings. In the proposed study, we will further characterize the effects of HIV protease inhibitors on the localization of these tight junction molecules. Gene expression levels will be assessed by protein and mRNA measurements. Gene transcription rate, mRNA stability, and promoter activity will be also investigated in both the HCAEC monolayer and pig carotid artery perfusion culture models. 3. Identify the signal transduction pathways involved in HIV protease inhibitor-induced endothelial permeability changes. We hypothesize that HIV protease inhibitors may: 1) alter the phosphorylation levels of tight junction proteins, and 2) induce increased endothelial permeability through signal transduction pathways involving ROS, MAPKs, protein kinase C (PKC), protein tyrosine kinase (PTK), and protein tyrosine phosphatase (PTPh). Again, our preliminary results provide support for this hypothesis. In the proposed study, we will use both the HCAEC monolayer and porcine carotid artery perfusion models to investigate the phosphorylation of occludin, ZO-1 and actin. We will measure the production of ROS, and assess the roles of protein kinases and PTPh in the observed permeability changes. In summary, these studies will help identify and establish new mechanisms of HIV protease inhibitor-induced endothelial permeability changes, thereby improving our understanding of the mechanisms of HAART-associated cardiovascular disorders. Our findings may also suggest new therapeutic strategies, which may eventually prove effective for the prevention and treatment of these complications.